Phenylalanine ammonia-lyase and ethylene in relation tochilling injury as affected by fruit age in citrus

Marıa T. Lafuente a,*, Lorenzo Zacarias a, Miguel A. Martınez-Tellez b,Marıa T. Sanchez-Ballesta a, Antonio Granell c

a Instituto de Agroquımica y Tecnologıa de Alimentos (IATA), Consejo Superior de Investigaciones Cientıficas (CSIC),

Apartado de Correos 73, Burjassot 46100, Valencia, Spainb Centro de Investigacion en Alimentacion y Desarrollo, A.C., CIAD, Apartado Postal 1735, Hermosillo, Sonora 83000, Mexico

c Instituto de Biologıa Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Cientıficas (CSIC),

Universidad Politecnica de Valencia, Avda Tarongers, s/n Valencia, Spain

Received 24 September 2002; accepted 11 March 2003

Abstract

Fruit of many citrus cultivars become injured when exposed to low, non-freezing temperatures. In this study we have

determined changes in ethylene production and phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in fruit of three citrus

cultivars, ‘Fortune’ mandarins, and ‘Navelina’ and ‘Valencia’ late oranges, with different tolerance to chilling injury

(CI) and demonstrated the influence of fruit physiological stage on those stress responses. We have shown that the

increase in ethylene production and PAL are cold-induced responses which are only stimulated in fruit of citrus

cultivars showing chilling damage and that both responses may occur concomitantly with the development of chilling

symptoms. However, the magnitude of these responses was not indicative of the degree of tolerance of a specific cultivar

to chilling. The influence of fruit age on both responses was evaluated in the most (‘Navelina’) and the least (‘Fortune’)

chilling tolerant cultivars. Chilling damage was not developed in ‘Navelina’ fruit at any physiological stage, but our

results in ‘Fortune’ mandarins, which always developed chilling symptoms, indicated that the induction of PAL in

response to chilling was dependent on the fruit physiological stage. Interestingly, increases in both PAL mRNA and

activity were barely affected by cold stress in the youngest ‘Fortune’ fruit harvested in December in spite of its

noticeable CI. For a similar CI index, the older the fruit, the higher was the shift in the levels of PAL transcript and in

PAL activity in response to cold. In contrast, the cold-induced ethylene production was little affected by the

physiological stage of the fruit.

# 2003 Elsevier B.V. All rights reserved.

Keywords: Citrus fruit; Chilling tolerance; Ethylene; Low temperature; Phenylalanine ammonia-lyase; Maturity

1. Introduction

Chilling injury (CI) is responsible for substantial

postharvest losses in many citrus cultivars. Chil-

ling induces pitting, necrosis and staining in the

* Corresponding author. Tel.: �/34-96-390-0022; fax: �/34-

96-363-6301.

E-mail address: postco@iata.csic.es (M.T. Lafuente).

Postharvest Biology and Technology 29 (2003) 308�/317

www.elsevier.com/locate/postharvbio

0925-5214/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0925-5214(03)00047-4

flavedo tissue (the outer coloured part of the peel)of fruit of these cultivars.

Physiological and molecular responses to low

temperature are not well understood. Temperature

extremes and a variety of environmental factors,

including irradiation, wounding, hypoxia, heavy

metals, waterlogging, drought, bending, disease

and insect attacks are able to induce ethylene

biosynthesis (Yang and Hoffman, 1984) and it iswell known that chilling induces an increase in

ethylene production in citrus fruit (Cooper et al.,

1969; McCollum and McDonald, 1991) and that

the cold-induced ethylene production may parallel

the increase in phenylalanine ammonia-lyase

(PAL) activity of cold-stored citrus fruit (Martı-nez-Tellez and Lafuente, 1997). In turn, it has been

shown that exogenous ethylene (Riov et al., 1969)or stresses that may favour ethylene biosynthesis,

such as wounding (Ismail and Brown, 1979) and

gamma radiation (Riov et al., 1968), may stimulate

PAL activity in the flavedo.

PAL is the initial rate-controlling enzyme in the

phenylpropanoid pathway. It is a key enzyme in

the phenolic metabolism that has been reported to

protect plants against stress conditions via differ-ent phenylpropanoid products (Hahlbrock and

Scheel, 1989; Dixon and Paiva, 1995). On the

other hand, the ethylene-induced increase in PAL

activity has been related to the development of

brown necrotic tissue areas in iceberg lettuce

(Hyodo et al., 1978). Inhibition of ethylene bio-

synthesis or action may prevent CI in horticultural

crops (Ben-Amor et al., 1999) but also favour it(Lafuente et al., 2001). A large body of literature

on the chilling responses of different fruit to 1-

MCP, an inhibitor of ethylene perception, is now

established in an updated web site http://

www.hort.cornell.edu/department/faculty/watkins/

ethylene/.

Previous results from our group indicate that

the induction of PAL and ethylene during coldstorage of ‘Fortune’ mandarin fruit may play a

role in reducing the development of chilling

symptoms and that the activation of PAL may

be dependent on ethylene but also an independent

cold signal apparently related to the cold-induced

peel damage (Lafuente et al., 2001). Whether PAL

or ethylene may serve as biological markers for

chilling sensitivity in citrus fruit or whether theability of citrus to cope with chilling stress is

directly related to its capacity to induce both

responses deserves further investigation.

In melon fruit, Diallinas and Kanellis (1994)

showed that the ripening stage may affect changes

in PAL and expression of genes for ethylene

biosynthesis in response to other stress conditions,

such as wounding. Young fruit showed a higherpotential to increase PAL protein content than

ripe fruit in response to wounding; whereas in

unripe melons the expression of ethylene biosyn-

thetic genes was not stimulated significantly by

wounding. The rise in PAL activity in response to

wounding or inoculation was also affected by fruit

ripening in bananas (Kamo et al., 2000). In

previous work we have demonstrated that thechilling susceptibility of ‘Fortune’ mandarins

changed during the season (Lafuente et al., 1997;

Holland et al., 2000). However, the influence of

fruit maturity on the cold-induced changes in PAL

activity and ethylene production has not been

examined.

Our research goals in the present work were to

determine whether: (1) the increase in ethyleneproduction and PAL are common responses to

chilling in fruit of citrus cultivars (‘Navelina’,

‘Valencia’ late, and ‘Fortune’) showing different

tolerance to this stress; (2) the constitutive levels of

PAL transcript or activity, or the ability of citrus

cultivars to increase PAL or ethylene production

in response to cold stress could be indicative of the

tolerance of a specific citrus cultivar to chilling;and (3) the cold-induced PAL activity and ethylene

production were affected by the fruit physiological

age.

2. Material and methods

2.1. Plant material and chilling treatments

Orange fruit of the cultivars ‘Navelina’ (Citrus

sinensis L. Osbeck) and ‘Valencia’ late (C. sinensis

L. Osbeck), and fruit of ‘Fortune’ mandarin

(Citrus clementina Hort. Ex Tanaka �/ Citrus

reticulata , Blanco) were harvested from trees

grown at Valencia, Spain (latitude: 39828?48ƒN;

M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317 309

longitude: 00822?52ƒW), all of them at commercialmaturity. ‘Navelina’ fruit were harvested in De-

cember, those of ‘Valencia’ late in April and

‘Fortune’ mandarins in March. In the two follow-

ing seasons, ‘Navelina’ or ‘Fortune’ fruit were

harvested from the same orchards at different

physiological ages, from November to February

or from December to April, respectively. At each

harvest time, three replicates of ten fruit wereselected to evaluate changes in fruit colour, size

and maturity index. An additional number of fruit

were divided at random into two groups for each

experiment and citrus cultivar. The first group was

used to determine changes in PAL activity and

ethylene production. Three replicates of ten fruit

per temperature and storage period for PAL

analysis and of three to four fruit for ethyleneproduction measurement were included in this

group. Flavedo samples were collected from the

total surface of fruit to evaluate the changes in

PAL gene expression and PAL activity, frozen in

liquid N2 and stored at �/80 8C until analysis. The

second group contained three replicates of 20 fruit

to determine chilling-induced peel damage.

Chilling stress was applied by exposing ‘For-tune’ mandarin fruit to 2 8C and 80�/85% relative

humidity (RH) for at least 28 days, and ‘Valencia’

late and ‘Navelina’ oranges to 2 8C and 80�/85%

RH for up to 60 days in constant darkness.

Control fruit were kept at 12 8C under the same

conditions.

2.2. Determination of surface area, peel colour and

maturity index

Fruit height and diameter were measured to

determine fruit surface area according to Turrell

(1946). Peel colour was determined using a Hunter

Lab Meter at four locations around the equatorial

plane of the fruit. Hue angle: 08�/red purple,

908�/yellow, 1808�/bluish-green, 2708�/blue. So-

luble solids (8Brix) and acids content of the juicewere measured to determine the fruit internal

maturity index, which was calculated by dividing

the 8Brix by its acid content. The content of

soluble solids was determined with an Atago/X-

1000 refractometer. Acids content was titrated

with 0.1 N NaOH using phenolphthalein as

indicator and expressed as g of anhydrous citric

acid in 100 ml of juice.

2.3. Estimation of CI index

Fruit were visually scored to estimate the extent

of CI development. Brown pit-like depressions of

the fruit are the main symptoms of CI. A rating

scale from 0 to 3, based on necrotic surface and

intensity of browning, was used to evaluate CI and

the average CI index determined as indicated in

the following formula: CI index�/a(CI scale (0�/

3)�/number of fruit in each class)/total number of

fruit estimated. The results are means of three

replicate samples containing 20 fruit9/S.E.M.

2.4. Assay of PAL activity

PAL (EC 4.3.1.5) activity was measured as

described by Martınez-Tellez and Lafuente

(1997) in three replicate samples from flavedo

acetone powder. Flavedo tissue samples were

periodically collected from the total surface of

fruit stored at 2 and 12 8C and ground in chilled

(�/20 8C) acetone. Ten ml of acetone were used per

g of flavedo. The homogenate was filtered, washed

twice with chilled acetone and the resulting powder

dried at room temperature. PAL was extracted

from 0.4 g of acetone powder with 15 ml of 0.1 M

sodium borate buffer, pH 8.8, containing 0.02 M

b-mercaptoethanol. Proteins were salted out with

ammonium sulphate to a final saturation of 46%

supernatant and thereafter dissolved in 4.5 ml of

0.1 M ammonium acetate buffer, pH 7.7, contain-

ing 0.02 M b-mercaptoethanol. PAL activity was

determined by measuring the absorbance of cin-

namic acid at 290 nm over a period of 2 h at 40 8Cand expressed on a dry-matter basis as nanomoles

of cinnamic acid per gram of acetone powder

flavedo tissue per hour. The reaction mixture

contained 2 ml of the purified enzyme extract

and 0.6 ml L-phenylalanine 0.1 M in a total

volume of 6 ml. Results are means of three

replicate samples9/S.E.M.

M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317310

2.5. RNA extraction and Northern analysis

Total RNA was isolated from 4 g of flavedo

tissue by the method of Cathala et al. (1983). Eight

mg of RNA were denatured at 65 8C and separated

on 1.2% agarose/formaldehyde gels and RNA

loading checked on ethidium bromide-stained

gels. The RNA was transferred to a Hybond-N�

nylon membrane (Amersham, Pharmacia,Biothech, Freiburg, Germany) using 20�/SSC

for at least 15 h and cross-linked using a UVC

500 Crosslinker (Amersham Pharmacia Biotech,

San Francisco, CA, USA). Filters were hybridised

at 65 8C in 7% (w/v) SDS, 0.33 M phosphate

buffer (pH 7.2) and 1 mM EDTA (pH 8). A

Fortune flavedo Fpal2 cDNA (Sanchez-Ballesta et

al., 2000a) was used as probe. Probes were labelledwith [a-32P]dCTP by the random primer labelling

method. Membranes were washed twice in 2�/

SSC, 0.1% SDS at room temperature, and twice

in 0.1�/SSC, 0.1% SDS at 65 8C, and exposed to

Kodak X-Omat SX film with intensifying screens

at �/80 8C.

2.6. Measurement of ethylene production

Three replicate samples of four ‘Fortune’ man-

darin fruit were incubated in 1 l glass jars, or of

three ‘Valencia’ late or ‘Navelina’ orange fruit in

1.5 l glass jars, for 4 h at 2 or 12 8C to determine

the ethylene production in chilled (2 8C) or non-

chilled control (12 8C) fruit. Three replicate sam-

ples of 1 ml gas sample were withdrawn from thehead space of each jar and injected in a gas

chromatograph equipped with a flame ionisation

detector. An activated alumina column was used.

Results are means of three replicate samples9/

S.E.M.

3. Results

3.1. Changes in PAL activity and ethylene

production during cold exposure of fruit from citrus

cultivars with different tolerance to CI

As shown in Fig. 1, ‘Fortune’ mandarins devel-

oped more CI than ‘Valencia’ late or ‘Navelina’

oranges. No pitting or rind staining was observed

in fruit of any of these citrus cultivars stored at

12 8C (data not shown). ‘Fortune’ fruit were stored

at 2 8C (chilling temperature) for up to 28 days

since severe damage occurred after this time. The

two other cultivars showed a much greater toler-

ance to chilling and were stored for up to 60 days.

After this period, we did not continue the experi-

ment since senescence of control fruit kept at 12 8Cbecame important. CI appeared by 14 days storage

at 2 8C on ‘Fortune’ mandarins. The CI index of

fruit of this cultivar was 1.1 and 2.7 by 14 and 28

days cold exposure, respectively. However, in

‘Valencia’ late oranges, slight peel damage (CI

index 0.4) was observed after 42 days storage. Peel

damage was not induced in fruit of the ‘Navelina’

cultivar even after 60 days storage at 2 8C (Fig. 1).

This behaviour was confirmed in further experi-

ments.PAL activity increased concomitantly with the

development of chilling-induced peel damage dur-

ing storage of ‘Fortune’ mandarins (Fig. 2A). The

activation of PAL in ‘Valencia’ late oranges also

Fig. 1. Changes in CI index of ‘Fortune’ mandarin (m) and

‘Valencia’ late (j) and ‘Navelina’ orange fruit (') held at 2 8C.

No CI was detected in fruit of these cultivars stored at 12 8C.

‘Fortune’ mandarins were stored for up to 28 days and

‘Valencia’ late and ‘Navelina’ oranges for up to 60 days.

Results of CI index are means of three replicate samples of 20

fruit9/S.E.M.

M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317 311

paralleled the development of CI (Fig. 2B). In this

cultivar, the cold-induced PAL activity increased

after 42 days (Fig. 2B) and was considerably lower

than that induced in ‘Fortune’ mandarins after 28

days exposure at 2 8C (Fig. 2A). In the most

chilling-tolerant cultivar (‘Navelina’), PAL activity

was similar to that of ‘Fortune’ mandarins and‘Valencia’ late oranges in freshly harvested fruit,

but it did not increase in response to the chilling

temperature (Fig. 2C). A similar trend in the cold-

induced ethylene production was found in fruit of

these cultivars (Fig. 2). Negligible differences

between the ethylene production of ‘Navelina’

fruit stored at the chilling and non-chilling tem-

perature were observed (Fig. 2C). As in the case ofPAL, the increase in ethylene production paral-

leled the development of chilling-induced peel

damage in ‘Fortune’ mandarins and ‘Valencia’

late oranges (Fig. 2A, B). The amount of ethylene

produced by ‘Valencia’ late oranges in response to

chilling was, however, higher than that of the least

chilling tolerant cultivar.

3.2. Influence of fruit age on cold-induced responses

‘Navelina’ oranges did not show CI or an

increase in PAL activity or ethylene production

during low temperature storage (60 days at 2 8C)

independently of the fruit physiological age (data

not shown). However, the tolerance of ‘Fortune’

mandarins to CI changed during the season (Fig. 3(season 1) and Fig. 4 (season 2)). Changes in size

and colour, as well as in the maturity index of fruit

of this cultivar are shown in Table 1 as indicators

of the external and internal degree of fruit

physiological age. The induction of PAL activity

in response to chilling increased with fruit age

(Figs. 3 and 4). In the first experiment in which we

evaluated the effect of fruit physiological stage(Fig. 3), we found that the effect of cold stress in

increasing PAL activity was clearly higher in fruit

harvested in February than in those harvested in

January although they showed a similar CI index

over the whole storage period. By 28 days at 2 8C,

PAL activity in fruit from January was about 30%

that of fruit from February. In fruit harvested in

December, PAL activity was barely affected bycold-stress despite CI being noticeable (Fig. 3). In

the following season, it was confirmed that, for a

similar CI index, the cold-induced PAL activity

was higher in fruit harvested later in the season

(Fig. 4). After 28 days storage at 2 8C, PAL

activity of fruit from April was about six times

Fig. 2. Changes in PAL activity (j, I) and ethylene produc-

tion (', ^) of ‘Fortune’ mandarin fruit (A), ‘Valencia’ late (B)

and ‘Navelina’ oranges (C) held at 2 8C (j, ') or 12 8C (I,

^). ‘Fortune’ mandarins were stored for up to 28 days and

‘Valencia’ late and ‘Navelina’ oranges for up to 60 days. PAL

activity was determined in flavedo tissue of fruit. Values of PAL

activity are means of three replicate samples of 10 fruit9/S.E.M.

and those of ethylene production means of three replicate

samples of three to four fruit9/S.E.M.

M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317312

that of fruit from December despite the initial

activity being similar (Fig. 4).

To investigate whether the effect of fruit phy-

siological age on the cold-induced PAL activity is

linked to the accumulation of PAL transcript,

total mRNA extracted from flavedo tissue from

freshly harvested ‘Fortune’ fruit and from fruit

stored for 21 days at 2 8C was analysed by

Northern hybridisation using a Fpal2 cDNA

probe. As shown in Fig. 5, the age at which the

fruit were harvested had a clear effect on the cold-

induced PAL mRNA accumulation in the flavedo.

As in the case of PAL activity (Fig. 3), no

difference was found before cold storage in the

levels of PAL transcript in fruit harvested during

the season, whereas the accumulation of PAL

mRNA in response to chilling increased with fruit

age. It is to be noted that no increase in transcript

levels occurred in fruit harvested in December.

In the following citrus season (Fig. 4), we

demonstrated that ethylene production in response

to cold stress was, however, barely affected by fruit

age. Ethylene production from freshly harvested

fruit was always very low (0.9�/1.5 pmol g�1 h�1).

Fig. 3. Changes in CI index and in PAL activity in ‘Fortune’

mandarins harvested during season 1 and exposed to chilling

stress. Fruit were harvested in December (m), January (j), and

February (') and held at 2 8C for up to 28 days. Values of CI

index are means of three replicate samples of 20 fruit9/S.E.M.

Results of PAL activity are the means of three replicate samples

of ten fruit9/S.E.M.

Fig. 4. Influence of fruit age on CI index, PAL activity (nmol

g�1 h�1) and ethylene production (pmol g�1 h�1) of ‘Fortune’

mandarins harvested during season 2 and exposed for up to 42

day at 2 8C. Values of CI index are means of three replicate

samples of 20 fruit9/S.E.M. Results of ethylene production are

means of three replicate samples of four fruit9/S.E.M. and

those of PAL activity means of three replicate samples of ten

fruit9/S.E.M.

M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317 313

Cold-stress always increased ethylene production,

as measured at 2 8C, but the rate of ethyleneproduction of fruit from January and February

was higher than that of fruit from April. Fruit

harvested in April showed a similar chilling

tolerance and a higher cold-induced PAL activity

than that of fruit harvested earlier in the season

(January and February) (Fig. 4). However, the

increase in ethylene production in fruit harvested

in December was similar (42 days) or higher (28days) than that of the more mature fruit from

April. This behaviour was further confirmed in the

following citrus season.

4. Discussion

In a previous work we found that constitutive

levels of PAL in a mandarin cultivar which showed

high tolerance to chilling were higher than those in

a mandarin showing very low tolerance (Sanchez-

Ballesta et al., 2000b). This result could suggest

that PAL serves as a biological marker for chilling

sensitivity in citrus fruit. However, we have found

in the present study that constitutive levels of PAL

activity in the flavedo of three other citrus

cultivars do not correlate with their tolerance to

chilling. Therefore, these results indicate that high

constitutive levels of PAL in the flavedo at harvest

are not indicative of chilling tolerance of different

citrus species. We have also shown that ethylene

production and PAL activity are common cold-

induced physiological responses only stimulated in

fruit of those citrus cultivars, which developed

chilling symptoms (Figs. 1 and 2) and that both

responses occurred concomitantly with the appear-

ance of symptoms. Interestingly, the least tolerant

cultivar showed the earliest and most pronounced

increase in ethylene production and PAL activity.

However, the magnitude of these responses was

not indicative of the degree of tolerance of a

specific cultivar to chilling. Thus, for a similar CI

index, the cold-induced increase in ethylene pro-

duction or PAL activity was higher in ‘Valencia’

late than in ‘Fortune’ fruit. It is to be noted that

stress-induced changes in PAL and ethylene de-

scribed in this experiment may be due to differ-

ences in the age of the peel of the fruit (Diallinas

and Kanellis, 1994; Kamo et al., 2000). These

cultivars were harvested at commercial maturity,

as determined by the ratio of total soluble solids/

acidity of the juice, since citrus are non-climacteric

fruit and there is not a good physiological marker

Table 1

Seasonal changes in fruit size, peel colour, and maturity index (soluble solids (8Brix)/acids content (g citric acid per 100 ml juice)) of

‘Fortune’ mandarin fruit harvested in two consecutive seasons

Season Month Fruit surface area (cm2) Colour index (h8) Maturity index

1 December 96.469/1.97 76.949/3.26 4.279/0.14

January 104.149/1.34 43.269/1.12 6.469/0.21

February 108.289/0.94 38.269/1.16 8.629/0.16

2 December 97.209/1.66 68.649/2.87 3.789/0.17

January 106.049/0.92 44.919/0.81 5.579/0.11

February 114.079/0.59 37.989/0.76 7.539/0.26

April 122.099/1.91 37.209/1.21 10.519/0.33

Values are means of three replicate samples of ten fruit9/S.E.M.

Fig. 5. Changes in PAL mRNA levels in ‘Fortune’ mandarins

harvested during season 1 and stored for 21 days at 2 8C. Fruit

were harvested in December (D), January (J), and February (F).

Eight micrograms of total RNA extracted from flavedo tissue

was fractionated, blotted, and hybridised with Fpal2 probe.

Equivalence of RNA loading was demonstrated by ethidium

bromide staining.

M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317314

which allows us to select different citrus cultivarswith the same peel physiological stage (Alonso et

al., 1995). Therefore, these results could not rule

out the influence of peel fruit age on the different

behaviour of the citrus cultivars examined in this

experiment. It is to be noted that ‘Navelina’

oranges did not show CI or an increase in PAL

activity or ethylene production during storage at

2 8C independently of the fruit physiological age(data not shown), while ‘Fortune’ mandarins (the

least tolerant cultivar) developed CI at any phy-

siological stage examined (Figs. 3 and 4).

PAL may be induced during development and

ripening in some climacteric (Diallinas and Ka-

nellis, 1994; Assis et al., 2001) and non-climacteric

fruit (Given et al., 1988). Our results in ‘Fortune’

mandarins indicating that PAL activity does notchange with the fruit physiological stage (Figs. 3

and 4) are in agreement with those reported by

Lisker et al. (1983) in grapefruit. Furthermore, we

have demonstrated that changes in PAL mRNA

levels (Fig. 5) paralleled those in PAL activity in

fruit harvested during the season (Fig. 3).

Increased PAL activity and ethylene production

have been reported to occur in citrus to protectfruit from CI (Lafuente et al., 2001). One of the

objectives of the present paper was to determine

whether the participation of the cold-induced PAL

and ethylene in protecting citrus fruit against

chilling were dependent on fruit age and, if so,

whether these responses were related to the chilling

susceptibility of the fruit. Since ‘Fortune’ mandar-

ins always developed chilling symptoms, changesin CI index, PAL levels and ethylene production

during cold storage of fruit of this cultivar

harvested during the seasons were compared.

During a first citrus season (Fig. 3) we observed

that the susceptibility to CI of fruit harvested in

January and February was similar and higher than

that of fruit harvested earlier in the season

(December). However, the older the fruit, thehigher was the shift in the cold-induced PAL

activity. Interestingly, the activity of the enzyme

was barely affected by cold stress in fruit harvested

in December despite of CI being noticeable.

Development of PAL activity in citrus can be

repressed by phenols (Dubery, 1990). In addition,

de novo synthesis of a PAL-inactivating factor has

been suggested to be involved in the loss ofethylene-induced PAL activity in lettuce (Ritenour

and Saltveit, 1996). Changes in PAL mRNA levels

in response to cold stress (Fig. 5) were tightly

linked to changes in PAL activity in ‘Fortune’ fruit

harvested at different physiological stages (Fig. 3).

Data shown in the present paper correspond to

fruit harvested after colour break. However, this

result was confirmed in other experiments usingfruit harvested from November (before colour

break) until April (data not shown). Therefore,

the effect of fruit age on the cold-induced changes

in PAL activity could be due to transcript levels

and does not need to invoke any PAL-inactivating

factors.

Considering these results and previous results

from our group indicating that the cold-inducedethylene production and PAL activity may be

related to the severity of cold-induced peel damage

(Sanchez-Ballesta et al., 2000a; Zacarıas et al.,

2002), we further investigated the effect of fruit age

on cold-induced PAL activity and whether or not

changes observed in PAL activity paralleled those

in ethylene production in the following citrus

season (Fig. 4). The chilling tolerance of ‘Fortune’mandarins harvested during this season was higher

than that of the first citrus season, probably

because their different preharvest daily tempera-

ture history (Gonzalez-Aguilar et al., 2000). In

order to reach a CI index comparable to that of

season 1, we prolonged the storage period until 42

days in season 2. It was confirmed that the ability

of fruit to increase PAL activity in response to coldstress increased with fruit age. Thus, PAL activity

in ‘Fortune’ fruit harvested in December and

stored for up to 42 days at 2 8C was about 24%

that of fruit from April (Fig. 4). However, the

cold-induced ethylene production appeared to be

little affected by the age of the fruit. The lack of

correlation between the rate of increase in both

responses in fruit harvested at different physiolo-gical stages demonstrates that ethylene may be a

triggering factor for PAL but other factors may

also contribute to the changes in PAL activity

occurring in the flavedo in response to chilling.

This would support the idea that cold-induced

PAL activity in citrus fruit may have different

inducers, dependent and independent on ethylene

M.T. Lafuente et al. / Postharvest Biology and Technology 29 (2003) 308�/317 315

(Lafuente et al., 2001). Interestingly, in grapefruitit was found that the ethylene-induced PAL

activity in the flavedo portion of the peel was

higher in the more developed fruit and that

treating the fruit with ethylene did not induce

PAL activity as long as the fruit were not

completely yellow (Lisker et al., 1983). In response

to other stress conditions, such as mechanical

wounding, controversial results have been foundsince PAL activity may be dependent but also

independent on ethylene production (Ke and

Saltveit, 1989; Diallinas and Kanellis, 1994). In

addition, the increases in ethylene production and

PAL activity have been shown to be independent

responses to pathogenic inoculation (Boller, 1991;

Meravy et al., 1991).

To our knowledge, this is the first reportshowing the influence of fruit physiological age

on the induction of PAL activity and on changes

in mRNA levels in response of citrus fruit to cold

stress. The increase in PAL activity in response to

other stress conditions, such as wounding or

inoculation with conidia, appeared to be also

influenced by fruit ripening. Thus, the rise in

PAL activity in wounded and/or inoculated ripebanana fruit was lower than that in unripe fruit

(Kamo et al., 2000). Ripe melon fruit showed also

a lower increase in PAL activity than unripe fruit

in response to wounding. In this climacteric fruit,

PAL gene expression followed the kinetics of

expression of the ethylene biosynthetic genes

during fruit development. In contrast, ethylene

biosynthetic genes were scarcely induced in youngunripe fruit but significantly activated in ripe fruit

by wounding (Diallinas and Kanellis, 1994).

The results reported in the present paper add

new information to our understanding of the role

of PAL in CI of citrus fruit and its relationship

with ethylene. Previous results indicate that the

induction of PAL in the cold-exposed ‘Fortune’

mandarins could be a protective response of thefruit to repair the damage originated by chilling

(Lafuente et al., 2001). Other results appeared to

suggest that the accumulation of PAL transcript

could serve as a molecular marker for chilling

tolerance in citrus fruit (Sanchez-Ballesta et al.,

2000b). From the results reported here, PAL still

stands as a defensive response of citrus against

chilling stress. Nevertheless, taking into considera-tion the data presented in this work, PAL induc-

tion appears not to be directly related to the

chilling tolerance of different citrus fruit. This

response was even not induced in fruit harvested

earlier in the season showing chilling symptoms.

Therefore, PAL appears not to be a good bio-

chemical marker for chilling tolerance in citrus

fruit. By contrast, chilling-induced ethylene waslittle affected by the fruit physiological stage.

In summary, from the overall results obtained in

this work we can conclude that: (1) the induction

of PAL activity and ethylene production occurred

concomitantly with the appearance of CI in the

chilling-sensitive citrus cultivars; (2) the increase in

PAL activity and PAL mRNA levels, but not in

ethylene, stimulated by CI is affected by the age ofthe fruit.

Acknowledgements

This work was supported by research grants

ALI-93-117 and ALI-96-0506-CO3 from the CI-

CYT, Spain and by FAIR-CT98-4096 from theEU. The technical assistance of D. Arocas is also

acknowledged.

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